SRC-Inhibition delays VEGF-Induced vascular endothelial cadherin (VE Cadherin) phosphorylation in endothelial cells

Abstract

Introduction. We previously demonstrated that VEGFR2 mediates VEGF-induced tyrosine phosphorylation of VE cadherin in endothelial cells. We hypothesize that VEGF-induced VE cadherin phosphorylation is regulated by Src tyrosine kinase. Methods. Human umbilical vein endothelial cells, HUVEC, were exposed to vehicle controls or VEGF (50 ng/ml) for 5–30 min, in the presence or absence of the Src inhibitor, PP2. KDR or VE cadherin were immunoprecipitated and probed with anti-phosphotyrosine or anti-VE cadherin using monoclonal antibodies to phosphotyrosine or VE cadherin. Experiments were performed in triplicate and data were analyzed by ANOVA. Results. VEGF caused early and transient tyrosine phosphorylation of VEGFR2 within 5 min of VEGF exposure (data not shown). In parallel, VEGF also caused early tyrosine phosphorylation of VE cadherin within 5 min, which decreased by 30 min (upper panel, figure below). Src inhibition using PP2 delayed the time course of VE cadherin phosphorylation, causing maximum VE cadherin phosphorylation at 30 min (upper panel). We also identified a KDR-VE cadherin complex in cells exposed to vehicle control. Conclusion. Endothelial signaling during VEGF exposure involves tyrosine phosphorylation of VE cadherin. Regulation of VEGF-induced VE cadherin phosphorylation appears to be Src-dependent.